The information provided herein and references cited are provided solely to assist the understanding of the reader, and does not constitute an admission that any of the references or information are prior art to the present invention.
With the explosion of techniques for the synthesis, analysis and manipulation of nucleic acids, numerous new opportunities have arisen in diagnostics and therapeutics. In research there is substantial interest in being able to identify DNA sequences, which may be associated with specific organisms, alleles, mutations, and the like, to understand particular genetic processes, to identify diseases, for forensic medicine, etc. Also, for many purposes, one may wish to modulate the expression of a target gene, so as to identify the function of such gene, or the cellular changes brought about by changes in the expression of such gene. In therapeutics, one may wish to inhibit the proliferation of cells, such as bacterial, fungal and chlamydia cells, which may act as pathogens, of viruses, of mammalian cells, where proliferation results in adverse effects on the host, or other situations. In vivo, one may provide for reversible or irreversible knock out, so that information can be generated on fetal development, or the effect on the organism of reduced levels of one or more genetic products.
Polyamide oligomers of nitrogen-containing five-membered heterocycles can be used to bind predetermined sequences of double stranded DNA (dsDNA). DNA recognition by polyamide oligomers depends on specific acid pairings that are oriented in the amino to carboxyl direction with respect to the 5′-3′ direction of the DNA helix. Thus, polyamide oligomers bind dsDNA in an antiparallel fashion and in a stoichiometric ratio of 1:1 or 1:2, oligomer to DNA (Dervan et al., Current Opinion in Chemical Biology, Vol. 3: 688, 1999). Antiparallel pairs of certain five-membered heterocycles preferentially bind to specific base pairs on duplex DNA. These antiparallel pairs have proven useful for the recognition of hundreds of predetermined DNA sequences by polyamide oligomers. Listed below in Table 1 are representative polyamide pairs of five-membered heterocycles and the DNA pairs that they preferentially bind to, referred to herein as the “pairing rules.”
TABLE 1Pairing Rules for Five-Membered HeterocylesPolyamide Pair*DNA base pair recognitionIm/PyG · CPy/ImC · GHp/PyT · APy/HpA · T*Im = N-methyl imidazole; Py = N-methyl pyrrole; Hp = 3-hydroxypyrrole
The fidelity of minor groove recognition by N-terminal Im/Py pairings in hairpin polyamides can be rationalized by a combination of both stabilizing and destabilizing forces which favors the rotamer with N3 in the groove and N-methyl out. Rotation of a terminal Im residue in the opposite conformer, orienting N3 away from the minor groove, would create unfavorable lone pair interactions with the proximal carboxamide oxygen, disrupt a favorable hydrogen bond with the exocyclic amine of G, and project an N-methyl group to the DNA floor which is presumably sterically unfavorable. Specifically, Im/Py distinguishes G-C from C-G and both of these from T-A/A-T base pairs while a Py/Py pair binds both T-A and A-T in preference to G-C/C-G. The exocyclic amino group of guanine imparts G-C specificity to Im/Py pairs through formation of a specific hydrogen bond with N3 of Im. Binding of Py/Py is disfavored at G-C base pairs by destabilizing steric interactions between the C3-H of Py and the guanine amino group (White et al., Chem. Biol. 1997, 4, 569; Kielkopf et al., Nat. Struct. Biol. 1998, 5, 104). The replacement of C3-H of one Py with hydroxyl creates the Hp/Py pair which exploits the steric fit and hydrogen bond acceptor potential of thymine-O2 as well as the destabilizing steric interaction with the bulkier adenine ring to gain specificity for T-A (White et al., Nature 391, 468, 1998; Kielkopf et al., Science 282, 111, 1998).
The five-membered heterocycles described thus far in DNA-binding polyamide oligomers are analogues of the pyrrole ring. Their chemical design mimics the natural products netropsin and distamycin A, molecules which bind the minor groove of DNA (Arcamone, F. et al., Nature 203: 1064, 1964; Pelton, J. G. et al., Proc Natl Acad Sci USA 86: 5723-5727, 1989).
The above pairing rules have been used to design hundreds of synthetic ligands that bind predetermined DNA sequences. However, many sequences remain difficult to target, likely due to sequence dependent microstructure variations in minor groove width or curvature. Furthermore, the specificity of cofacial aromatic amino acid pairings depend on their context (position) within a given hairpin polyamide. For example, Im/Py pairings show comparable specificity for G-C at both terminal and internal positions. Conversely, Hp/Py pairings do not specify T-A at the N-terminus of hairpin polyamides (Ellervik et al., J. Am. Chem. Soc. 2000, 122, 9354). The context dependence of Hp is presumably a result of the conformational freedom inherent to an N-terminal aromatic residue. The absence of a second ‘groove-anchoring’ carboxamide allows terminal rings to bind DNA in either of two conformations. For a terminal Hp residue, a rotamer with the hydroxyl recognition element oriented away from the floor of the minor groove could be stabilized by intramolecular hydrogen bonding between the C3-OH and the carbonyl oxygen of the 2-carboxamide. For terminal 2-hydroxybenzamide residues, some measure of T-A selectivity was recovered by creating steric bulk at the 6-position to force the hydroxyl recognition element into the groove (Ellervik et al., J. Am. Chem. Soc. 2000, 122, 9354). However, N-terminal pairings capable of binding T-A, with affinity and specificity comparable to those of Im/Py for G-C, are desired.
Efforts have been devoted to extend the ensemble of five-membered heterocycles that are capable of cooperatively pairing with each other to recognize specific DNA base pairs. These efforts have, in part, been motivated by the instability of the Hp heterocycle towards acids and free radicals. Polyamide oligomers containing Hp are susceptible to such degradation, and a robust replacement for use in biological applications is desired.
A search for new five-membered heterocycles and new five-membered heterocycle pairs for sequence determination was recently attempted with little success (Marques, M. et al., Helvetica Chimica Acta 85: 4485-4517, 2002). Using molecular modeling from an X-ray crystallography structure of a polyamide oligomer bound to duplex DNA, analogs of the existing five-membered heterocycles were designed to optimize binding to the curvature and twist of minor-groove DNA. Analogs of Py (1-methyl-1H-pyrazole (Pz) and 1H-pyrrole (Nh)), Im (5-methylthiazole (Nt) and furan (Fr)), and Hp (3-hydroxythiopene (Ht)) were synthesized and investigated in polyamide pairs. Additional sulfur containing pyrrole analogs (4-methylthiazole (Th), 3-methylthiophene (Tn), and thiophene (Tp)) were also studied. The chemical structures of these analogs are shown below:

Six-membered heterocycles represent a new class of heterocycles that may be employed in compounds which bind DNA. Certain small molecule ligands known to bind the minor groove of DNA with relatively high affinities contain six-membered heterocycles and fused heterocycles, such as benzimidazole, imidazopyridines, and indoles (R. L. Lombardy, et al., J. Med. Chem., 39: 1452-1462, 1996; Minehan, T. G. et al. Helv. Chim. Acta, 83: 2197-2213, 2000; Wang, L. et al., Biochemistry, 40: 2511-2521, 2001; Zhang, W. et al., FEBS Lett., 509: 85-89, 2001; Ji, Y.-H. et al., Bioorg. Med. Chem., 9: 2905-2919, 2001; Satz, A. L. et al., J. Am. Chem. Soc., 123: 2469-2477, 2001; Behrens, C. et al., Bioconjugate Chem, 12: 1021-1027, 2001; Matsuba, Y. et al., Cancer Chemother. Pharmacol., Vol. 46: 1-9, 2000).
Hoechst 33258, which comprises a bis-benzimidazole, an N-methylpiperazine, and a phenol moiety, is an example of a fused six-membered cyclic derivative (P. E. Pjura, K. Grzeskowiak, R. E. Dickerson, J. Mol. Biol., 197: 257-271, 1987; M. Teng, N. Usman, C. A. Frederick, A. Wang, Nucleic Acids Res., 16: 2671-2690, 1988; S. Kumar, B. Yadagiri, J. Zimmermann, R. T. Pon, J. W. Lown, J. Biomol. Struct. Dyn., 8: 331-357, 1990). The chemical structure of Hoechst 33258 is shown below:

Hoechst 33258 is a highly fluorescent dye which binds the minor groove of DNA at A•T rich tracks. Oligomers of the Hoechst benzimidazoles have been synthesized and studied for DNA recognition (Minchan, T. G. et al., Helvetica Chimica Acta 83: 2197-2213, 2000). These benzimidazole oligomers also show preference for A•T rich sequences, as well as for 5′-WGWWW-3′ and 5′-WCWWW-3′, where W=A or T.
While Hoechst 33258 and its corresponding benzimidazole oligomers bind A•T rich DNA in a 1:1 ratio, such compounds do not recognize specific nucleotide base pairs across the duplex, such as in the pairing rules described above for five-membered heterocyclic polyamide oligomers. Other six-membered heterocyclic DNA binding ligands reported thus far also do not recognize specific mononucleotide base pairs across the duplex.
Thus, a need in the art for a T recognition element using the asymmetric cleft of a T-A base pair as the basis for shape selective discrimination has been identified.